Figure 1.
The calcineurin inhibitor FK506 blocks hyphal growth and enforces multi-budded yeast growth in M. circinelloides.
(A) On YPD agar media, eight M. circinelloides isolates (upper: CBS277.49, ATCC1216b, R7B, and ATCC1216a, bottom: NRRL3631, ATCC1207b, NRRL1443, ATCC1207a) grow as hyphae, whereas in the presence of FK506 (1 µg/mL), they form a compact yeast colony. (B) In liquid YPD media with vigorous shaking for aeration, wild-type exhibits hyphal growth. When FK506 is present in the media, Mucor grows only as a multi-budded yeast, as is also observed during growth under low oxygen/high CO2 conditions (isolate grown at the bottom of a flask completely filled with liquid YPD media generating microaerobic/high CO2 conditions). Representative images with isolate CBS277.49 are shown here. Similar results were observed with other Mucor isolates (data not shown). Scale = 5 µm.
Figure 2.
Conserved domains of the three catalytic A subunits of M. circinelloides.
(A) The calcineurin catalytic A subunits of M. circinelloides, R. delemar, and P. blakesleeanus have a highly conserved N-terminal phosphatase catalytic domain followed by a unique C-terminal region with domains mediating binding to 1) the calcineurin B regulatory subunit; 2) cyclophilin A-CsA complex; 3) FKBP12-FK506 complex (depicted as blue box and in B panel); and 4) calmodulin (yellow box), and an auto-inhibitory domain (orange box) that docks into the active site. This C-terminal regulatory domain distinguishes calcineurin from all other known protein phosphatases. (B) The calcineurin B subunit binding domain is in red font. Asterisk is for identical amino acids, colon is for conservative amino acid substitutions, and period is for semiconservative amino acid substitutions. Hs: Homo sapiens, Sc: S. cerevisiae, Cn: C. neoformans, An: A. nidulans, Mc: M. circinelloides, Rd: R. delemar, and Pb: Phycomyces blakesleeanus.
Figure 3.
Phenotype of the spontaneous mutants that are resistant to FK506 and sensitive to rapamycin and CsA.
(A and B) The four mutants MSL11 (CNBR-1), MSL12 (CNBR-2), MSL15 (CNBR-1), and MSL16 (CNBR-3) each have a mutation in the latch region of the cnbR gene (also see Figure S4) and the mutants formed mycelia when the calcineurin inhibitor FK506 (1 µg/mL) is present in the media (The CNBR-3 mutant is shown in Figure S6). The CNBR-1 and CNBR-2, and CNBR-3 mutants are still sensitive to rapamycin. The CNBR-1 and CNBR-2 mutants remain sensitive to CsA, whereas CNBR-3 was found to be CsA cross-resistant (Figure S5 and S6). The fkbAΔ mutant and L91P mutants, by contrast, are resistant to FK506 and rapamycin due to the absence or alteration of FKBP12, the target for both drugs, respectively. (C and D) The MSL13 (CNAA-1) and MSL14 (CNAA-2) mutants have mutations in the cnaA gene (see also Figure S7), resulting in amino acid alterations in the binding domains for calcineurin B and for the FKBP12-FK506 complex. Similarly to the CNBR-1, and CNBR-2, the CNAA-1 and CNAA-2 mutants form hyphae in the presence of FK506 but remain sensitive to rapamycin and CsA.
Table 1.
Strains and plasmids used in this study.
Figure 4.
cnbRΔ mutants lacking the calcineurin B regulatory subunit are locked in yeast phase growth.
(A) On YPD solid media, the cnbRΔ mutants lacking all calcineurin activity (CnbR is the common subunit partner for the three catalytic subunits CnaA, CnaB, and CnaC) produce a small compact colony; however, wild-type forms a complex mycelial mat. (B) In liquid media with vigorous shaking for aeration, wild-type grows as a filamentous/hyphal fungus, whereas the cnbRΔ mutants only exhibit multi-budded yeast growth as observed in wild-type grown in the presence of FK506 or anaerobic/high CO2 conditions. (C) The two cnbRΔ mutants were serially diluted and spot-inoculated on YPD or YPD with FK506 (1 µg/L). FK506 treatment did not impact growth of the mutants. Scale = 20 µm.
Figure 5.
Yeast-locked mutants are attenuated in virulence in a heterologous host model.
(A) 20,000 wild-type spores, wild-type yeast, or the cnbRΔ yeast-locked mutants were inoculated into wax moth larvae. Infection with wild-type spores or yeast resulted in 100% mortality by day 4 post-infection; however, the cnbRΔ yeast-locked mutants were significantly less virulent compared to wild-type spores or yeast cells. (B) At an inoculum of 40,000 cells, the cnbRΔ mutants exhibited 60 to 70% mortality by day 2 post-infection, whereas wild-type spores showed 100% mortality.
Figure 6.
Protein kinase A activity is elevated during yeast growth.
(A) Bicarbonate ions (HCO3−) that are equivalent to a high concentration of CO2 induce yeast growth of Mucor. Scale = 20 µm. (B) Crude protein extract (0.5 µg) from each sample was subjected to a PKA-specific kinase activity measurement with the SignaTECT cAMP-Dependent Protein Kinase (PKA) Assay System (Promega, Madison, WI). Protein kinase A activity was elevated during yeast growth phase induced by CO2, and similar results were observed during yeast growth enforced by FK506 or in the cnbRΔ yeast-locked mutants, indicating that higher PKA activity is correlated with yeast growth and calcineurin may negatively regulate PKA activity. Error bars represent the standard deviation of the mean from three independent assays (from left to right, the values are: 3.06±3.01; 15.04±12.99; 11±4.6; 13.82±9.16).
Figure 7.
Three cna genes in M. circinelloides are differentially expressed.
(A) The three cna gene are differentially expressed during yeast and hyphal growth. A difference in intensity of the cnaC gene RT-PCR product was apparent when yeast induced by low oxygen/high CO2 or FK506 were compared with hyphae. (B) Expression of the cnaC gene increased ∼3-fold during yeast growth compared to hyphal growth; however, expression of both cnaA and cnaB decreased during yeast growth based on qRT-PCR analyses. The actin gene served as a normalization control, and the hyphal growth stage was used as a control to measure the expression of the cna genes during yeast growth.
Figure 8.
cnaAΔ mutants are sensitive to calcineurin inhibitors and SDS.
(A) Two independently derived cnaA mutants exhibit hypersensitivity to FK506 or CsA. In the presence of low concentrations of FK506 (0.1 µg/L), growth of the cnaAΔ mutants was significantly impaired compared to the wild-type, which formed a readily visible colony after only 1 day. (B) In liquid culture with a low concentration of FK506 (0.1 µg/mL), at which wild-type grows as hyphae, the cnaAΔ mutants grew as multi-budded yeast cells. Even in culture without FK506, the cnaAΔ mutants exhibited a mixture of yeast and hyphae compared to wild-type, which was exclusively hyphal. Scale = 20 µm. (C) The cnaAΔ mutants were more sensitive to SDS in YPD media than wild-type, suggesting that the cnaAΔ mutants fail to have compromised cell wall integrity. The percentage concentration (v/v) of SDS is displayed in the panel.
Figure 9.
cnaAΔ calcineurin A subunit mutants produce more virulent and larger spores than wild-type.
(A) The cnaAΔ mutants and wild-type produced multinucleate spores (stained with DAPI), and the mutant spores were larger than those of wild-type. DIC: differential interference contrast, DAPI: nuclei. (B) Wild-type produced spores with a size of 12.0±2.95 µm, whereas the cnaAΔ1 and cnaAΔ2 strains produced significantly larger spores with a size of 16.1±4.04 or 16.0±3.70 µm, respectively. The difference in spore size between wild-type and the cnaAΔ mutants was statistically significant (p<0.0001, N = 80). (C) The larger spores of the cnaAΔ mutants were more virulent than wild-type at the inocula tested (N = 5,000), suggesting that calcineurin is involved in spore size determination and providing further evidence that spore size is linked to the virulence of this fungus.
Figure 10.
Hyphal growth is altered in cnaAΔ mutants.
(A) Wild-type spores first grow isotropically and then polarity is established to initiate germ tube emergence. During hyphal growth, polarity is maintained properly in order to grow directionally. (B) Compared to wild-type, the cnaAΔ mutants exhibited a shorter isotropic growth phase due to their larger initial spore size (the cnaAΔ mutants germinated at 50 minutes after inoculation, whereas wild-type started germination at 75 minutes after inoculation, as shown in the figure) [53]. cnaAΔ mutant hyphae undergo tip-splitting (solid red arrows), indicating that hyphal polarity is not properly maintained during mycelial growth. The cnaAΔ mutants also show an abnormal hyphal branching pattern (open red arrows). The numbers on the figure indicate the time (in minutes) after inoculation. Scale = 20 µm.
Figure 11.
Illustration of roles of calcineurin in dimorphism and virulence of Mucor.
Calcineurin is necessary for hyphal growth and negatively regulates spore size. Calcineurin is also involved in cell wall integrity. Calcineurin activity is inhibited by two immunophilin-drug complexes, FKBP12-FK506 and CypA-CsA, resulting in yeast growth or abnormal hyphal growth, respectively. On the other hand, during yeast growth, cAMP-dependent protein kinase A (PKA) activity is increased. I) Inhibition of calcineurin activity by FK506 or mutation of the regulatory B subunit results in elevated PKA activity, indicating that calcineurin negatively regulates PKA activity, either via direct inhibition of PKA or indirectly through other factor(s). II) Alternatively, PKA and calcineurin may share common target proteins and function. FKBP12: FK506 binding protein 12, CypA: cyclophilin A.